Tobacco use causes five million deaths worldwide annually and is the leading cause of preventable mortality in the Western world. Nicotine is the addictive component of tobacco that binds to and activates a family of ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs). Activation of the receptors in the dopaminergic (DAergic) mesolimbic reward pathway is thought to underlie the initiation of addiction. One of the difficulties in understanding nicotine's effect on nAChRs arises from the existence of multiple nAChR subtypes, each exhibiting unique electrophysiological properties and varying affinities for nicotine. Twelve distinct neuronal nAChR subunits have been identified (12-110 and 22-24). Five subunits co-assemble to form receptors with the subunit composition of each channel determining its pharmacological and biophysical properties. Chronic nicotine exposure alters the expression of nAChR subtypes, which likely contributes to nicotine dependence;however, the underlying mechanisms regulating these changes remain unclear. A growing body of evidence indicates that nicotine and cigarette smoke alters the expression of small 21-24 nucleotide long regulatory molecules, referred to as microRNAs (miRNAs). miRNAs are predicted to regulate over 60% of the human genome and the majority of all mammalian protein coding genes, typically by binding to complementary sites in the 3'-untranslated regions (3'-UTRs) of target mRNAs and guiding them to an RNA- induced silencing complex (RISC). A miRNA, miR-1, has been reported to target nAChRs in C. elegans, leading to changes in nAChR properties. We carried out bioinformatics analysis of mammalian 14 and 22 nAChR genes and found that they contain predicted binding sites for several known miRNAs. This proposal will investigate the hypothesis that nicotine-induced changes in miRNA expression mediate functional changes in the mesolimbic pathway (the reward pathway) by altering the expression of nAChR subtypes. To address this, I will use a combination of molecular biology, electrophysiology, and pharmacology. In AIM 1 I will examine if miRNAs regulate nAChR subunits 14 and 22. In AIM 2 I will look at nicotine's effect on the expression levels of miRNAs that function within the DAergic mesolimbic pathway. In AIM 3 I will test if miRNAs are necessary and/or sufficient to cause nicotine-induced changes in nAChR functional properties of DAergic neurons from the midbrain. Ultimately, gaining a better understanding of the molecular processes that act to control nicotine dependence-associated behaviors will lead to the development of novel therapies for smoking cessation. PUBLIC HEALTH RELEVANCE: Tobacco use is the second leading cause of death in the world and the single largest preventable cause of disease and premature mortality. Nicotine, the primary addictive component of tobacco, is among the most addictive substances of abuse. The goal of this project is to gain a better understanding of the molecular processes that act to control expression of the neuronal receptors for nicotine. We anticipate that insights gained from this project will help lead to the development of therapies for smoking cessation.